The formation of blood clots in blood vessels of major organs in the body is one of the leading causes of human mortality in Western industrialized society. Myocardial infarction--heart attack--primarily caused by blood clot formation or thrombosis in the coronary artery, is the leading cause of death in the United States among adult males. Emboli, blood clots traveling in the circulatory system, which lodge in the blood vessels of the lung, brain, or heart are also significant causes of death in patients following surgery, dialysis and traumatic injury. Phlebitis, a condition in which thrombi, stationary blood clots, block circulation through the large blood vessels, particularly of the lower extremities, is also a serious threatening disease.
The mechanism of blood clot dissolution or fibrinolysis is complex. At least three components are involved; plasminogen, plasminogen activators and plasmin inhibitors. Plasminogen is one of the circulating plasma proteins incorporated into a blood clot as it forms. Plasminogen is an inactive precursor or proenzyme form of the protein plasmin, a proteolytic enzyme that digests fibrin threads, as well as other substances involved in the activation of blood clot formation such as fibrinogen, factor V, factor VIII, prothrombin, and factor XII. Limited proteolysis of plasminogen yields plasmin. Plasminogen can be proteolytically activated to form plasmin by a number of enzymatically active proteins known as plasminogen activators. Plasminogen has a specific binding affinity for fibrin and thus a portion of the circulating plasminogen accumulates in the blood clot in association with the fibrin reticulum of the clot.
There are a number of commercially known plasminogen activators presently available for use in thrombolytic therapy including streptokinase, urokinase, recombinant tissue type plasminogen activator and acylated streptokinase-plasminogen. Streptokinase does not specifically bind to fibrin and as a result, it activates both circulating plasminogen and plasminogen in the blood clot. Two-chain urokinase is similar to streptokinase in its pattern of activity and the generalized, rather than local manner in which they exert their plasminogen activation, is a major drawback in therapeutic use.
Single-chain urokinase (scuPA) and tissue plasminogen activator (tPA) are fibrin-specific thrombolytic agents and are thus expected to cause less bleeding complications resulting from a systemic fibrinogenolysis at doses that are therapeutically effective. While these plasminogen activators are more fibrin-specific, tPA has a short half-life in the patient, on the order of two to five minutes (Matsuo, (1982) Throm Haemostas 48:242) and the half-life of scuPA is similarly limited (Stump et al (1987) J Pharm Exp Therap 242(1):245-250).
Modification of these therapeutically useful plasminogen activators to increase the half-life while maintaining desired biological activities of the activators would allow the use of these activators in the mammalian fibrinolytic system in lower dosages to achieve comparable thrombolytic efficacy with the concomitant potential advantages of reduced proteolysis of plasma proteins, prevention of reocclusion for longer periods, and reduced production cost per therapeutic dose.
The problems of short half-life mentioned above and other undesirable properties of certain activators are well recognized and various modifications of the activators have been undertaken to solve them. These include the modification of tissue plasminogen activator (tPA) to prevent site specific N-glycosylation (Lau et al, (1987) Biotechnology 5:953-957) and identifying the function of the structural domains of tPA in order to construct second generation plasminogen activators with improved fibrinolytic activity (see for example, Klausner, (1986) Biotechnology 4:709-711 and references cited therein).
Clinical studies have shown that concurrent or subsequent administration of heparin with tPA therapy is recommended to keep blood clotting suppressed. Heparin is a conventional anticoagulant which is employed in conditions in which a rapid reduction in the coagulability of the blood is desired. The major disadvantage associated with heparin therapy is the principal toxic effect of hemorrhage.
The heparin molecule consists of (1-4)-linked 2-amino-2-deoxy-alpha-D-glucopyranosyl, alpha-L-idopyransyluronic acid and a relatively small amount of beta-D-glucopyransouyluronic acid residues.
Stassen et al, (1987) Thromb Haemostasis 58(3):947-950, describe the potentiation of thrombolysis by recombinant tPA and scuPA in the presence of high doses of two low molecular weight fractions of heparin.
Heparin has been bound covalently to solid supports to prepare blood-compatible surfaces. For example, PCT application WO86/03318 published 27 Dec. 1985 (Cardiol. Sci Center) discloses immobilization of urokinase on a heparin support to produce water-soluble complex with increased thrombolytic activity. The conjugation is via a carboxyl group, not an aldehyde group.
Paques et al, 1986 Thromb Res 42:797-807, describe an affinity complex formed between tPA or urokinase (uPA) and heparin. Apparently the heparin-binding site is related with the fibrin-binding site of these plasminogen activators.
It is desirable to conjugate the plasminogen activator to a smaller heparin fragment. Hoffman et al, (1983) Carbohydrate Res 117:328-331 discloses a method for producing heparin fragments using nitrous acid, wherein the fragments have 2,5-anhydro-D-mannose residues as reducing terminal units with aldehyde groups. Such aldehyde groups mays be reacted with primary amines to give labile Schiff-bases which can be converted to stable secondary amines by reductive amination. Hoffman et al describes coupling of such heparin fragments to Sepharose and curdlan, a (1.fwdarw.3)-linked unbranched beta-D-glucan, and suggests coupling of heparin to human serum albumins and antithrombin.
U.S. Pat. No. 4,745,180 describes pharmaceutical compositions composed of water-insoluble proteins such as recombinant forms of beta-interferon, interleukin-2 or certain immunotoxins that are conjugated to at least one heparin fragment to produce a product which is water soluble.
None of the references, however, disclose how to use heparin fragments to increase the half-life of plasminogen activators nor teach the targeting of heparin to the critical site of clot dissolution. Furthermore, it is not a priori possible to predict which selected plasminogen activators would be favorable responsive, e.g., have good biological activity, to treatment with heparin fragments due to physical and pharmacokinetics differences among the plasminogen activators on the one hand, and albumins, cytotoxins and lymphokines, on the other hand.
There have been reports in the literature that administration of erythropoietin (EPO) to some users has a tendency to increase the sedimentation level of the blood and this has led to complications and death of some users of EPO. Also, EPO in its unglycosylated form as provided in bacterial host systems, has a relatively short half-life making it unsuitable for human clinical use. Accordingly, EPO is currently manufactured in relatively expensive mammalian cell host systems so that the resulting protein is glycosylated. The glycosylated form has a sufficient half-life to be useful clinically. There is no prior art showing how to use heparin fragments to increase the half-life of EPO, especially unglycosylated EPO, or to use heparin to prevent clotting as a result of increased sedimentation level of the blood of a person administered with EPO.